Identification of novel functional sites in the Ca v 1.3 calcium channel α1-subunit using evolutionary modeling

Figures

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  Fig. 1.. Evolutionary pathogenicity predictions overlap with established functional and structural sites of Cav1.3. Cartoon representation of domain I of the Cav1.3 α1-subunit, comprising voltage-sensing domain I (VSD I) and pore domain I (PD I). Zoomed regions highlight residues with known functional relevance: (A) epistatic score matrix, where darker blue indicates lower scores and higher predicted pathogenicity, lighter blue to orange indicates higher scores and lower pathogenicity, and white with a central dot denotes wild-type (WT); (B) residues forming the structural basis for ion selectivity; (C) residues of the charge transfer center; (D and E) residues mediating interdomain interactions between S4 and S5, and between the S4–S5 linker and S5; and (F) residues involved in hydrophobic gating. Backbone coloring reflects evolutionary algorithm–derived pathogenicity scores shown in (A), with darker colors indicating more negative scores, higher context-dependent conservation, and increased likelihood of deleterious substitutions. For visualization, residues with scores above −4.6 (low predicted pathogenicity) are shown in white. Side chains are colored by physicochemical class: polar…

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  Fig. 2.. Pathogenicity predictions based on our evolutionary model outside the classical functional sites. Cartoon illustration of VSD and PD of the Cav1.3 α1-subunit. Residues are highlighted in zoom (boxes): (A) R1401 participates in multiple polar contacts between PLs (I and IV) and the extracellular loop (IV); (B) A376 connects pore loop (PL) to the rest of the pore domain (PD); (C) F1362 mediates the coupling in the S4–S5 interface (between IV S5 and III S4); (D) S635 establishes connection between II S4–S5 linker and III S5 in the activation gate region; (E) Q1156 determines the correct movement of III S6 relative to II S6 in response to the change in the channel state; (F) N745 participates in the interaction network surrounding the AG region. The color of the backbone represents the score obtained using the same evolutionary based algorithm as in Fig. 1. The side chains are colored according to the type of residues shown in Fig. 1.

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  Fig. 3.. Structural and functional consequences of the S635I variant (domain II, S4–S5 linker). (A–C) shows that S635I induces a leftward shift of activation (WT: n = 24; S635I: n = 23), enhanced steady-state inactivation (WT: n = 21; S635I: n = 16) and increased current density (WT: n = 24; S635I: n = 23). (D) shows the position of S635I in domain II. The structural illustration of the mutation effect in (E) shows that S635I establishes an additional hydrophobic contact with the activation gate region at the bottom of the II S6 helix in the preopen state, facilitating channel opening at lower voltage. In the open state, mutation to apolar isoleucine (in green) disrupts a stabilizing polar contact between S635 (in salmon) and N1017 in III S5, facilitating transition to the inactivated state. The side chains are colored according to the type of residues shown in Fig. 1.

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  Fig. 4.. Structural and functional consequences of the F1362A variant (domain IV, S5). (A–C) The F1362A mutation induces a rightward shift in the voltage-dependent activation (WT: n = 16; F1362A: n = 17) and steady-state inactivation (WT: n = 13; F1362A: n = 9) together with slowed inactivation (WT: n = 16; F1362A: n = 16). (D) marks the position of F1362A in the S4–S5 interface. Structural illustration of the mutation effect in (E) shows that F1362A loosens the coupling in the S4–S5 interface by disrupting the hydrophobic and polarizable contacts. The side chains are colored according to the type of residues shown in Fig. 1.

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  Fig. 5.. Structural and functional consequences of the A376V variant (domain I, pore loop). (A–C) The A376V induces a leftward shift in the voltage-dependent activation (WT: n = 24; A376V: n = 23) and steady-state inactivation (WT: n = 7; A376V: n = 9) along with accelerated inactivation (WT: n = 18; A376V: n = 13). (D) marks the position of A376V near the pore loop (PL) and the interaction network between PLs and those connecting PL to the rest of the pore domain (PD) in domain I. The structural illustration of the mutation effect in (E) shows that A376V replaces the minimal side chain of alanine with a much larger hydrophobic valine. This improves the PL–PD binding by strengthening the hydrophobic contact with M297 in II PL, while inducing distortion of the PL from its native conducting conformation. The side chains are colored according to the type of residues shown in Fig. 1.

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  Fig. 6.. Structural and functional consequences of the R1401A variant (domain IV, pore loop). (A–C) R1401A induces a leftward shift in the voltage-dependent activation (WT: n = 24; R1401A: n = 21). (D) marks the position of R1401A in the interface between pore loops (PLs), near the selectivity filter and connecting the extracellular loop in domain IV. The structural illustration of the mutation effect in (E) shows that R1401A alters multiple polar contacts between PLs and to the extracellular loop. The side chains are colored according to the type of residues shown in Fig. 1.

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  Fig. 7.. Structural and functional consequences of the N745A variant (domain II, S6). (A) marks the position of N745A and the interaction network surrounding the AG region detailed in D). (B and C) The N745A results in a nonconducting channel (WT: n = 24; N745A: n = 16). (D) provides structural illustration of the mutation effect that shows that N745A disrupts several contacts in the preopen state. The side chains are colored according to the type of residues shown in Fig. 1.

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  Fig. 8.. Structural and functional consequences of the Q1156E variant (domain III, S6). (A–C) The Q1156E enhances inactivation (WT: n = 22; Q1156E: n = 20) and reduces the number of noninactivated channels (WT: n = 21; Q1156E: n = 18). (D) marks the position of Q1156E in the channel structure (domain III). The structural illustration of the mutation effect in (E) shows that Q1156E substitutes an optimal hydrogen bond with electrostatic repulsion from E759. This change helps to move the distal III S6 into the ion path during inactivation. The side chains are colored according to the type of residues shown in Fig. 1.